Matrix-assisted laser desorption/ionization (MALDI) is a method allowing for ionization of chemical compounds. Usually, it is used together with a time-of-flight (TOF) mass analyzer and is widely used for mass spectrometric analysis of chemicals. Because of wide range of analytes that can be analyzed and short analysis time, the MALDI-TOF mass spectrometric technique is widely used for structural analysis of various solid materials, particularly biomolecules.
However, because of very poor reproducibility of MALDI mass spectral patterns, it is difficult to use MALDI mass spectrometry for quantitative analysis of analytes. For this reason, the industrial or scientific applications of MALDI mass spectrometry are very limited.
For quantitative analysis of an analyte using MALDI mass spectrometry, various MALDI mass spectrometric techniques have been developed, including relative quantification without using an internal standard, relative quantification using an internal standard, absolute quantification using an internal standard, and absolute quantification using an analyte added.
The relative quantification without using an internal standard (or profile analysis) is a MALDI mass spectrometric method wherein a classification algorithm is used for reproducible analysis of MALDI mass spectra based on the fact that the relative signal intensity of each component in the MALDI mass spectra is constant. However, the weakness of the profile analysis method is that the design and practice of experiments are difficult.
The relative quantification using an internal standard is a MALDI mass spectrometric method wherein an analyte is quantified by measuring the peak height or area of each analyte in the MALDI mass spectra of samples to which a predetermined amount of an internal standard has been added relative to the peak height or area of the internal standard. However, with the relative quantification method using an internal standard, the absolute amount of the analyte cannot be determined.
The absolute quantification using an internal standard is a MALDI mass spectrometric method wherein a calibration curve is constructed from several samples containing different amount of an analyte to be measured as well as a constant amount of an internal standard, and the absolute amount of the analyte is determined from the calibration curve based on the relative amount of the analyte obtained from an unknown sample according to the relative quantification method using an internal standard described above. However, the absolute quantification method using an internal standard is disadvantageous in that a calibration curve has to be constructed for each component if a sample containing multiple components is to be analyzed.
The absolute quantification using an analyte added is a MALDI mass spectrometric method wherein a sample containing an analyte to be analyzed is divided into two or more samples, calibration points are obtained from the MALDI mass spectra obtained for the samples containing different amounts of the analyte, and the absolute amount of the analyte is determined from the calibration points. However, the absolute quantification method using an analyte added has the problem that the analyte to be analyzed needs to be prepared additionally and several samples are needed for the analysis of one analyte.
The currently known methods for quantitative analysis using MALDI mass spectra use an internal standard, particularly a compound identical to the analyte but substituted with an isotope. However, when the analyte has a large molecular weight, such as proteins, nucleic acids, etc., or when the degree of isotopic substitution is increased to distinguish the mass spectrum of the analyte substituted with the isotope from that of the unsubstituted analyte, the cost increases greatly. Another disadvantage of the MALDI mass spectrometry-based quantitative analysis using an internal standard is that the analyte pretreatment is not simple.
Since the sample in MALDI mass spectrometry is usually a mixture of an analyte and a matrix, an analyte ion (AH+) and fragmentation products thereof and a matrix ion (MH+) and fragmentation products thereof appear in the MALDI mass spectrum. Accordingly, the MALDI spectral pattern is determined by the fragmentation patterns of AH+ and MH+ and the ratio of the intensities of AH+ and MH+.
The ions generated by MALDI can be fragmented inside (in-source decay, ISD) or outside (post-source decay, PSD) the ion sources. The ISD occurs and terminates fast, whereas the PSD occurs slowly. The rate and yield of the fragmentation reaction of the analyte ion are determined by the reaction rate constant and the internal energy of the ion. Accordingly, if the effective temperature of a plume generated by a laser pulse in MALDI is known, the internal energy can be determined and the reaction rate can be calculated therefrom.
There have been many scientific researches to find out the temperature of a plume, which is a gas containing ions and neutral molecules generated when a laser is irradiated on a sample in MALDI mass spectrometry (J. Phys. Chem. 1994, 98, 1904-1909; J. Am. Soc. Mass Spectrom. 2007, 18, 607-616; J Phys. Chem. A 2004, 108, 2405-2410).
However, the most systematic method for measuring the plume temperature was first presented by the inventors of the present disclosure (J. Phys. Chem. B 2009, 113. 2071-2076). The inventors of the present disclosure have succeeded in obtaining the ion fragmentation reaction rate and effective temperature through kinetic analysis of time-resolved photodissociation spectra and PSD spectra. The obtained temperature was found to be the late plume temperature (Tlate). The inventors of the present disclosure could also determine the early plume temperature (Tearly) by analyzing the ISD yield using a reaction rate function obtained therefrom.
First, the inventors of the present disclosure measured the intensities of the fragmented ion products of peptide ions generated by ISD, PSD, etc. from MALDI spectra. From the data, the survival probabilities (Sin) of the peptide ions at the ion source exit were calculated. The maximum rate constant at which the peptide ions can survive at the ion source exit was obtained in consideration of the experimental conditions and the maximum internal energy corresponding thereto was determined from the fragmentation rate constant of the peptide ions. The internal energy distribution of the peptide ions was obtained while varying temperatures and Tearly, i.e. the temperature at which the probability of the region below the maximum internal energy is identical to Sin, was determined.
The early and late temperatures of the ion-containing gas (plume) determined by the method devised by the inventors of the present disclosure matched well with those reported previously by other researchers. However, the method of the inventors of the present disclosure is advantageous in that it is methodologically more systematic and more universally applicable due to the lack of randomness, when compared with the methods devised by other researchers (Journal of the American Society for Mass Spectrometry, 2011, vol. 22, pp. 1070-1078).
In addition, the inventors of the present disclosure have surprisingly found out that, although the early plume temperature (Tearly) varies if the MALDI experimental condition is changed, the mass spectral patterns of the spectrums with the same Tearly are identical even when the mass spectra are obtained under different experimental conditions (Korean Patent Application No. 10-2012-0075891).
The inventors of the present disclosure have found out that, if Tearly is the same, not only the mass spectral pattern but also the total number of generated ions (total ion count, TIC) is also the same. This suggests that mass spectra can be obtained at the same Tearly by maintaining Tearly by adjusting the energy intensity of a laser pulse irradiated to a sample.
In addition, the inventors of the present disclosure have found out that the reaction quotient of the proton exchange reaction of the plume (Q=[M][AH+]/([MH+][A])) obtained from the spectra having the same Tearly is constant for regardless of the change in analyte concentration in the solid samples. That is to say, the inventors of the present disclosure have found out that in MALDI-TOF mass spectrometry the early plume is almost in thermal equilibrium and the reaction quotient (Q) is equal to the equilibrium constant (K) of the proton exchange reaction between the matrix and the analyte. Accordingly, in MALDI-TOF mass spectra the ratio of the intensities of the analyte and matrix ions generated under a constant-temperature condition is directly proportional to the analyte-to-matrix molar ratio in the solid sample and quantitative analysis will be possible based thereon.
The inventors of the present disclosure have invented a method for measuring the equilibrium constant of an ionization reaction between a matrix and an analyte, wherein MALDI spectra are obtained at the same Tearly by adjusting the intensity of a laser pulse irradiated to a sample and the ratio of the signal intensity of the matrix ion and the signal intensity of the analyte ion is measured from the obtained MALDI spectra.
In addition, the inventors of the present disclosure have invented a method for obtaining a calibration curve for the change in the ratio of the concentrations of a matrix and an analyte at constant temperature using the equilibrium constant of the reaction between the matrix and the analyte.
Also, the inventors of the present disclosure have invented a method for quantitative analysis of measuring the amount of an analyte included in a sample prepared by mixing a predetermined amount of a matrix with an unknown amount of the analyte by calculating the moles of the analyte by substituting the ratio of the signal intensity of the analyte ion and the signal intensity of the matrix ion measured from the mass spectra of the sample as well as the concentration of the matrix into the calibration curve.